JPH0411194B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a method for producing 1-(4-methoxyphenyl)-2-aminopropane by an enzymatic method.

(Prior art) 1-(4-methoxyphenyl)-2 by enzymatic method
-Propanone to 1-(4-methoxyphenyl)-
A method for obtaining 2-aminopropane has been completely unknown so far.

(Structure and Effects of the Invention) 1-(4-methoxyphenyl)-2-aminopropane, which is the target compound of the present invention, is a carbostyril derivative (Japanese Patent Application Laid-Open No. 1989-1979-1), which is an excellent pharmaceutical compound having bronchodilatory action. No. 208965) is an important material as a raw material. The present inventors obtained this 1-(4-
As a result of repeated research on the production method of 1-(4-methoxyphenyl)-2-aminopropane, it was found that 1-(4-methoxyphenyl)-2-propanone was converted into 1-(4-methoxyphenyl)-2-propanone using microorganisms such as Brevibacterium. The present invention was completed based on the discovery that the present invention has the ability to specifically convert -2-aminopropane.

That is, the present invention provides 1-(4-methoxyphenyl)
- A culture solution of a Brevibacterium microorganism capable of producing 1-(4-methoxyphenyl)-2-aminopropane from 2-propanone, bacterial cells collected from the culture solution, or a processed product of the bacterial cells. 1
1-(4-methoxyphenyl)- produced by reacting with -(4-methoxyphenyl)-2-propanone
This is a method for producing 1-(4-methoxyphenyl)-2-aminopropane, which is characterized by collecting 2-aminopropane.

The Brevibacterium genus microorganism used in the present invention includes 1-(4-methoxyphenyl)-2-
1-(4-methoxyphenyl)-2 from propanone
- Any microorganism of the genus Brevibacterium that has the ability to produce aminopropane may be used, and specific examples include Brevibacterium linens IFO12141, Brevibacterium protophormiae IFO12128, and the like. Any strain of these microorganisms can be suitably used, such as a wild strain, a mutant strain, or a recombinant strain induced by a genetic method such as a cell fusion method or a genetic manipulation method.

The medium for culturing the above microorganisms is not particularly limited,
Any material that can be used for culturing microorganisms may be used. For example, as a carbon source, any carbon source that can be used by the microorganisms mentioned above can be used. Specifically, sugars such as glucose, fructose, sucrose, and dextrin, sugar alcohols such as glycerol and sorbitol, fumaric acid, and citric acid can be used as carbon sources. Organic acids such as acids can be used. The amount of these carbon sources added to the medium is preferably about 0.1 to 10%.
Examples of nitrogen sources include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium phosphate; ammonium salts of organic acids such as ammonium fumarate and ammonium citrate; meat extracts; yeast extracts; cornstarch liquor;
Natural organic nitrogen sources such as casein hydrolyzate can be used, and in many cases, the organic nitrogen source can also be used as a carbon source. The amount of the nitrogen source added is usually in the range of 0.1 to 10%.
Examples of inorganic salts include alkali metal phosphates such as potassium phosphate and sodium phosphate; alkali metal chlorides such as potassium chloride and sodium chloride;
Sulfate metal salts such as magnesium sulfate and ferrous sulfate can be suitably used, and the amount used is usually
A range of 0.001 to 1% is appropriate.

Cultivation of microorganisms at 20-40℃, especially 30-37℃, PH
Preferably it is carried out under aerobic conditions at a pH of about 5-8, especially about 6-7.

In addition, 1-(4-methoxyphenyl)-2 was added to the medium.
- Adding a small amount of aminopropane can increase the ability to produce the target compound of the present invention.
The proportion of 1-(4-methoxyphenyl)-2-aminopropane to be added varies slightly depending on the microorganism used, but it is usually 0.001% or more, especially 0.1%.
~1% is preferred. The added 1-(4-methoxyphenyl)-2-aminopropane can be used as a nitrogen source in culturing the above-mentioned microorganism.

The culture solution thus obtained and the microbial cells collected from the culture solution can be suitably used as an enzyme source. Furthermore, the bacterial cell-treated product (for example, freeze-dried bacterial cells, acetone-dried bacterial cells, washed bacterial cells, ground bacterial cells, autolysed bacterial cells, ultrasonicated bacterial cells, bacterial cell extracts, etc.) is treated with an enzyme. It can also be used as a source. In addition, the bacterial cells or treated bacterial cells can be immobilized and used by known methods such as polyacrylamide gel method, sulfur-containing polysaccharide gel method (carrageenan gel method, etc.), alginate gel method, agar gel method, etc. .
Furthermore, enzymes purified from bacterial cell extracts using a combination of known methods can also be used.

The reaction is carried out by mixing the culture solution, bacterial cells, or treated bacterial cells with 1-(4-methoxyphenyl)-2-propanone, which is a substrate. It may be carried out by adding a substrate to the culture solution containing the bacterial cells after culturing,
Alternatively, the bacterial cells obtained from the culture solution or the treated bacterial cells may be added to an aqueous solution of the substrate and reacted. The concentration of the substrate is preferably approximately 0.05 to 5%, particularly 0.1 to 1%. The pH of the reaction solution is preferably 5-10, particularly 6-9. The reaction temperature is generally 10~50℃, especially 25~
40°C is preferred.

Furthermore, if this reaction is carried out in the presence of an amino group donor, a surfactant, a coenzyme, etc., the reaction time can be shortened or the amount of 1-(4-methoxyphenyl)-2-aminopropane accumulated can be increased. It is preferable to be able to measure Examples of amino group donors used for this purpose include ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate,
Examples include ammonium salts such as ammonium acetate and ammonium formate, and L-amino acids such as L-glutamic acid and L-aspartic acid, and it is preferable to use them in equal molar or more to the substrate. Examples of surfactants include cetylpyrimidium bromide, cetyltrimethylammonium bromide, polyethylene glycol, p-isooctylphenyl ether (manufactured by Rohm and Haas, USA, trade name: Triton X-100), etc. For the reaction solution,
It is preferable to use about 0.0001 to 0.1%. Furthermore,
An example of the coenzyme is nicotinamide adenine dinucleotide (reduced type), which is preferably used in an amount approximately equimolar to the substrate.

1-(4-
Methoxyphenyl)-2-aminopropane can be easily collected as hydrochloride crystals by extracting with an organic solvent such as ethyl acetate, concentrating under reduced pressure, and acidifying with hydrochloric acid.

As mentioned above, the method of the present invention is an industrially inexpensive 1-
This is the first successful production of 1-(4-methoxyphenyl)-2-aminopropane, which is important as a raw material for pharmaceutical compounds, from (4-methoxyphenyl)-2-propanone by an enzymatic method. Optical purity can be achieved by selecting appropriate enzyme reaction conditions such as microorganisms.
This is an excellent method that can yield 100% product.

Hereinafter, the present invention will be explained in detail with reference to Examples.
In addition, in this specification, all "%" shall mean "weight/capacity (g/dl)."

Example 1 Glucose 0.5%, peptone 1%, meat extract 1
%, enzyme extract 1.25%, and sodium chloride 0.5% (PH 7.0) was placed in a 500 ml shake flask and sterilized at 120°C for 10 minutes. Brevibacterium protoformiae was added to this medium.
Inoculate one platinum loop of IFO12128 and culture with shaking at 30°C for 18 hours. 1-(4-methoxyphenyl)-2-
Propanone 4.9g, ammonium chloride 40.1g, 1M
- Suspend 300 ml of Tris-HCl buffer and 30 ml of 1% cetyltrimethylammonium bromide aqueous solution in a pre-dissolved substrate aqueous solution of approximately 2.5 (PH 8.0), and add water to make 3. By reacting this suspension at 30° C. for 2 days, (R)-1-(4-methoxyphenyl)-2-aminopropane is produced. Cells were removed by centrifugation, and 1% sodium hydroxide solution was added to the resulting supernatant to adjust the pH to 11.0, followed by extraction with ethyl acetate and drying under reduced pressure. After adding a small amount of ethyl acetate to dissolve and filtering off insoluble materials, isopropyl alcohol containing 10% hydrochloric acid was added and the precipitated crystals were collected by filtration to obtain (R)-1-(4-methoxyphenyl)- 0.4 g of 2-aminopropane hydrochloride is obtained.

[α] ²⁰ _D : -22.8° (C=0.5, H ₂ O) Optical purity: 100% Example 2 Brevibacterium linens IFO12141 was cultured in the same manner as in Example 1. The bacterial cells collected by centrifugation from culture solution 1 were mixed with 1.6 g of 1-(4-methoxyphenyl)-2-propanone, 13.4 g of ammonium chloride, 100 ml of 1M Tris-HCl buffer and 1
% cetyltrimethylammonium bromide aqueous solution 10ml
Suspend in approximately 800 ml of pre-dissolved substrate solution (PH8.0) and add water to make 1.

When this suspension is reacted at 30℃ for 2 days (S)
0.8 g of -1-(4-methoxyphenyl)-2-aminopropane is produced. Purification is carried out in the same manner as in Example 1 to obtain 0.7 g of (S)-1-(4-methoxyphenyl)-2-aminopropane.

[α] ²⁰ _D : +22.8° (C=2, H ₂ O) Optical purity: 100% Example 3 Brevibacterium linens IFO12141 is cultured in the same manner as in Example 1. Bacterial cells collected by centrifugation from 10 ml of culture solution were mixed with 16.4 mg of 1-(4-methoxyphenyl)-2-propanone, 134 mg of ammonium chloride, 70 mg of nicotinamide adenine dinucleotide (reduced form), and 1 ml of 1M Tris-HCl buffer. and 10 ml of substrate solution (PH
8.0).

By reacting this suspension at 30°C for 2 days, 16 mg of (S)-1-(4-methoxyphenyl)-2-aminopropane was obtained. (conversion rate 100%)

Claims (1)

[Claims] 1 A culture solution of a Brevibacterium microorganism having the ability to produce 1-(4-methoxyphenyl)-2-aminopropane from 1-(4-methoxyphenyl)-2-propanone, collected from the culture solution It is characterized by allowing bacterial cells or a processed product of bacterial cells to act on 1-(4-methoxyphenyl)-2-propanone and collecting the generated 1-(4-methoxyphenyl)-2-aminopropane. A method for producing 1-(4-methoxyphenyl)-2-aminopropane.